Activated sialic acid derivatives for protein derivatisation and conjugation

ABSTRACT

Derivatives of PSAs are synthesized, in which a reducing and/or non-reducing end terminal sialic acid unit is transformed into a N-hydroxysuccinimide (NHS) group. The derivatives may be reacted with substrates, for instance substrates containing amine or hydrazine groups, to form non-cross-linked/crosslinked polysialylated compounds. The substrates may, for instance, be therapeutically useful drugs, peptides or proteins or drug delivery systems.

The present application is a continuation of and claims priority to U.S.patent application Ser. No. 14/581,784, filed on Dec. 23, 2014, which inturn claims priority to U.S. patent application Ser. No. 14/162,856, nowU.S. Pat. No. 8,952,141, filed on Jan. 24, 2014, which in turn claimspriority to U.S. patent application Ser. No. 13/544,882, now U.S. Pat.No. 8,735,557, filed on Jul. 9, 2012, which claims priority to U.S.patent application Ser. No. 11/816,823, now U.S. Pat. No. 8,217,154,filed on Aug. 21, 2007, which is the national stage entry ofPCT/GB2006/000540, filed on Aug. 12, 2005, and also claims priority toEP 05251017.9, filed on Feb. 23, 2005, and PCT/GB2005/003160, filed onAug. 12, 2005. The entire contents of each of the above patentapplications are hereby expressly incorporated by reference.

The present invention relates to derivatives of compounds such aspolysialic acids, which have terminal sialic acid units, and preferablyconsisting essentially of only sialic acid units, having anN-hydroxysuccinimide (NHS) group for reaction with substrates at thereducing or non-reducing end and to methods of producing them. Thederivatives are useful for conjugation to amine-group containingsubstrates such as peptides, proteins, drugs, drug delivery systems(e.g. liposomes), viruses, cells (e.g. animal cells), microbes,synthetic polymers or copolymers etc.

Polysialic acids (PSAs) are naturally occurring unbranched polymers ofsialic acid produced by certain bacterial strains and in mammals incertain cells [Roth et. al., 1993]. They can be produced in variousdegrees of polymerisation: from n=about 80 or more sialic acid residuesdown to n=2 by either limited acid hydrolysis, digestion withneuraminidases or by fractionation of the natural, bacterially or cellderived forms of the polymer. The composition of different PSAs alsovaries such that there are homopolymeric forms i.e. the alpha-2,8-linkedPSA comprising the capsular polysaccharide of E. coli strain K1 and ofthe group-B meningococci, which is also found on the embryonic form ofthe neuronal cell adhesion molecule (N-CAM), Heteropolymeric forms alsoexist, such as the alternating alpha-2,8 alpha-2,9 linked PSA of E. colistrain K92 and the group C polysaccharides of N. meningitidis. Inaddition, sialic acid may also be found in alternating copolymers withmonomers other than sialic acid such as group W135 or group Y of N.meningitidis. PSAs have important biological functions including theevasion of the immune and complement systems by pathogenic bacteria andthe regulation of glial adhesiveness of immature neurons during foetaldevelopment (wherein the polymer has an anti-adhesive function)[Muhlenhoff et. al., 1998; Rutishauser, 1989; Troy, 1990, 1992; Cho andTroy, 1994], although there are no known receptors for PSAs in mammals.The alpha-2,8-linked PSA of E. coli strain K1 is also known as‘colominic acid’ and is used (in various lengths) to exemplify thepresent invention.

The alpha-2,8 linked form of PSA, among bacterial polysaccharides, isuniquely non-immunogenic (eliciting neither T-cell nor antibodyresponses in mammalian subjects) even when conjugated to immunogeniccarrier protein, which may reflect its existence as a mammalian (as wellas a bacterial) polymer. Shorter forms of the polymer (up to n=4) arefound on cell-surface gangliosides, which are widely distributed in thebody, and are believed to effectively impose and maintain immunologicaltolerance to PSA. In recent years, the biological properties of PSAs,particularly those of the alpha-2,8 linked homopolymeric PSA, have beenexploited to modify the pharmacokinetic properties of protein and lowmolecular weight drug molecules [Gregoriadis, 2001; Jain et. al., 2003;U.S. Pat. No. 5,846,951; WO-A-0187922], PSA derivatisation of a numberof therapeutic proteins including catalase and asparaginase [Fernandesand Gregoriadis, 1996 and 1997] gives rise to dramatic improvements incirculation half-life and their stability and also allows such proteinsto be used in the face of pre-existing antibodies raised as anundesirable (and sometimes inevitable) consequence of prior exposure tothe therapeutic protein [Fernandes and Gregoriadis, 2001]. In manyrespects, the modified properties of polysialylated proteins arecomparable to proteins derivatised with polyethylene glycol (PEG). Forexample, in each case, half-lives are increased, and proteins andpeptides are more stable to proteolytic digestion, but retention ofbiological activity appears to be greater with PSA than with PEG[Hreczuk-Hirst et. al., 2002]. Also, there are questions about the useof PEG with therapeutic agents that have to be administered chronically,as PEG is only very slowly biodegradable [Beranova et. al., 2000] andboth high and low molecular weight forms tend to accumulate in thetissues [Bendele, et. al., 1998; Conyers, et. al., 1997]. PEGylatedproteins have been found to generate anti PEG antibodies that could alsoinfluence the residence time of the conjugate in the blood circulation[Cheng et. al., 1990]. Despite, the established history of PEG as aparenterally administered polymer conjugated to therapeutics, a betterunderstanding of its immunotoxicology, pharmacology and metabolism willbe required [Hunter and Moghimi, 2002; Brocchini, 2003]. Likewise thereare concerns about the utility of PEG in therapeutic agents that requirehigh dosages, (and hence ultimately high dosages of PEG), sinceaccumulation of PEG may lead to toxicity. The alpha 2,8 linked PSAtherefore offers an attractive alternative to PEG, being animmunologically ‘invisible’ biodegradable polymer which is naturallypart of the human body, and that can degrade, via tissue neuraminidasesto sialic acid, a non-toxic saccharide.

Our group has described, in previous scientific papers and in grantedpatents, the utility of natural PSAs in improving the pharmacokineticproperties of protein therapeutics [Gregoriadis, 2001; Fernandes andGregoriadis, 1996, 1997, 2001; Gregoriadis et. al., 1993, 1998, 2000;Hreczuk-Hirst et. al., 2002; Mital, 2004; Jain et. al., 2003, 2004; U.S.Pat. No. 5,846,951; WO-A-0787922]. Now, we describe new derivatives ofPSAs, which allow new compositions and methods of production ofPSA-derivatised proteins (and other forms of therapeutic agents). Thesenew materials and methods are particularly suitable for the productionof PSA-derivatised therapeutic agents intended for use in humans andanimals, where the chemical and molecular definition of drug entities isof major importance because of the safety requirements of medical ethicsand of the regulatory authorities (e.g. FDA, EMEA).

Methods have been described previously for the attachment ofpolysaccharides to therapeutic agents such as proteins [Jennings andLugowski, 1981; U.S. Pat. No. 5,846,951; WO-A-0187922]. Some of thesemethods depend upon chemical derivatisation of the ‘non-reducing’ end ofthe polymer to create a protein-reactive aldehyde moiety (FIG. 1). Thereducing end of PSA (and other polysaccharides) is only weakly reactivewith proteins under the mild conditions necessary to preserve proteinconformation and the chemical integrity of PSA during conjugation. Thenon-reducing end of sialic acid terminal unit, which contains vicinaldiols, can be readily (and selectively) oxidised with periodate to yielda mono-aldehyde derivative. This derivative is much more reactivetowards proteins and comprises of a suitably reactive element for theattachment of proteins via reductive amination and other chemistries. Wehave described this previously in U.S. Pat. No. 5,846,951 andWO-A-0187922. The reaction is illustrated in FIG. 1 in which;

-   -   a) shows the reaction of the aldehyde with a primary amine group        of a protein after the oxidation of CA (alpha-2,8 linked PSA        from E. coli) with sodium periodate to form a protein-reactive        aldehyde at the non-reducing end of the terminal sialic acid and    -   b) shows the selective reduction of the Schiff's base with        sodium cyanoborohydride (NaCNBH₃) to form a stable irreversible        covalent bond with the protein amino group.

In WO2005/016973 we describe polysaccharide derivatives which have asulfhydryl-reactive group introduced via a terminal sialic acid unit.This unit is usually introduced by derivatisation of a sialic acid unitat the non-reducing end of the polysaccharide. The sulfhydryl reactivegroup is preferably a maleimido group. The reaction to introduce thisgroup may involve reacting a heterobifunctional reagent having asulfhydryl-reactive group at one end and a group such as a hydrazide oran ester at the other end with an aldehyde or amine group on the sialicacid derived terminal unit of the polysaccharide. The product is usefulfor site specific derivatisation of proteins, e.g. at Cys units orintroduced sulfhydryl groups.

Although the various methods that have been described to attach PSAs totherapeutic agents [U.S. Pat. No. 5,846,951; WO-A-01879221], aretheoretically useful, achievement of acceptable yields of conjugate viareaction of proteins with the non-reducing end (aldehyde form) of thePSA requires reaction times that are not conducive to protein stabilityat higher temperature (e.g. interferon alpha-2b). Secondly, reactantconcentrations (i.e. polymer excess) are required that may beunattainable or uneconomical.

Therefore, we have solved the above problems by developing a new methodfor conjugation of polysialic acids which have NHS-sialic acid groups atthe reducing and/or non-reducing termini, to proteins. The weakreactivity of the reducing end can be exploited to beneficial effect (bydestroying the non-reducing end, capping the reducing end andderivatising with a bifunctional crosslinker), thus avoiding the productcomplexity described in FIGS. 2 and 3 using the established method(FIG. 1) of reductive amination of proteins with periodate oxidised CA.

Jennings and Lugowski, in U.S. Pat. No. 4,356,170, describederivatisation of bacterial polysaccharides with proteins via anactivated reducing terminal unit involving a preliminary reduction stepfollowed by an oxidation step. Examples where this approach has beenemployed by Jennings et al include polysaccharides wherein the reducingterminal unit is N-acetyl mannosamine, glucose, glucosamine, rhamnoseand ribose.

In EP-A-0454898 an amino group of a protein is attached to an aldehydegroup which has been synthesised by reducing and partially oxidising thereducing terminal sugar moiety of a glycosaminoglycan. Theglycosaminoglycans treated in this way include hyaluronic acid,chondroitin sulphate, heparin, heparan sulphate, and dermatan sulphate.None of these compounds has a sialic acid unit at the reducing terminal.

In the invention there is provided a novel compound comprising apolysialic acid substrate having at least one of its terminal unitsderived from a sialic acid unit which includes an ester ofN-hydroxysuccinimide linked to the terminal unit at either the 2 or7-carbon, optionally via a linker. The N-succinimidyloxy group ishereinafter referred to as an NHS group. In this invention thesuccinimidyl moiety may be unsubstituted or substituted by groups suchas sulphonyl or other groups to confer useful solubility properties. Thederivatised terminal unit may be derived from a non-reducing terminalsialic acid group or from a reducing terminal sialic acid group. Theremay be two such NHS groups per PSA molecule, for instance one on aterminal unit derived from a non-reducing terminal sialic acid group andthe other derived from a reducing terminal sialic acid group.

The compounds of the invention may also be defined in terms of generalformulae. The novel compounds preferably have the general formula I, IIor III

-   -   in which R¹ is H or sulfonyl;    -   R² is a linking group;    -   A is NR⁵, NR⁵NR⁶, O or SR wherein R⁵ and R⁶ are independently        selected from H, C₁₋₄ alkyl and aryl;    -   SylO is a sialyl group;    -   n is 1-100 and m is 0-100;    -   R³ is hydrogen or a mono-, di-, oligo- or polysialic acid group,        a protein, a peptide, a lipid, a drug, a component of a cell        membrane or wall or a drug delivery system; and    -   R⁴ is hydrogen or a mono-, di-, oligo- or polysialic acid group,        an alkyl group, an acyl group, a drug or a drug delivery system.

The linking group, R², together with the NHS group and ester linkagewhich are found in compounds I to III typically form a structure whichis derived from the bifunctional NHS reagent used to synthesise thecompounds. Suitable bifunctional reagents are listed later in theapplication. During the synthesis the group labelled as A which isderived from a PSA starting material attaches itself to the appropriateend of the bifunctional reagent with corresponding loss of a leavinggroup from the NHS reagent (at an opposite end to the NHS group whichdoes not react), or alternatively structural rearrangement of thereagent. Typically, the linking group R² will comprise an alkane-diylgroup together with a carbonyl to which A is attached in compounds offormula I to III. Preferably R² is C_(p)H_(2p)CO where p is 2-12.Alternatively, the linking group may comprise an alkanediyl groupwherein one of the alkane carbon atoms is attached to the A group. R²may include mid-chain ester, amide, ether, thioether and/or1-thio-N-succinimidyl amine linkages for instance derived frompreliminary derivatisation reactions of a PSA reagent or of an NHSreagent. R² may be an alkyleneoxyalkylene group or analkyleneoligooxyalkylene group.

A is preferably NR⁵, wherein R⁵ is hydrogen, which is derived from aprimary amine PSA starting reagent. Examples of such amine PSAderivatives and methods for producing them are given below.

In one embodiment the terminal sialic acid unit has been subjected to apreliminary chemical reaction to generate useful functional groups towhich a maleimide-group containing reagent may be linked.

In one embodiment we have found it convenient to use the chemistrydisclosed in our earlier publications in which an aldehyde group isgenerated, as a preliminary step to generate the functional group viawhich the NHS group may be linked.

The invention includes processes for forming the novel compounds. Thereis also provided a new process for synthesising the new compounds inwhich a PSA substrate is reacted, optionally after preliminaryderivatisation step(s) of the terminal sialic acid unit(s), with abifunctional reagent, one of the functionalities of which is an NHSester and the other of the functionalities is reactive with a sialicacid unit(s) or derivative(s), thereof, as the case may be, underconditions such that covalent conjugation between the reagent and the 2or 7 carbon atom from the terminal sialic acid group(s) or derivativethereof occurs and the NHS group remains unchanged.

In a preferred embodiment the sialic acid unit in the substrate issubjected to a preliminary step in which an amine group is generated.

Where the sialic acid unit which is derivatised is a reducing terminalunit the preliminary step may involve amination at the anomeric carbon,or, preferably, the following sequence of steps:

-   -   a) reduction to open the ring of the reducing terminal sialic        acid unit to form a vicinal diol group;    -   b) selective oxidation of the vicinal diol group formed in        step a) to form an aldehyde group;    -   c) conversion of aldehyde in step b) to an amino group by        reductive amination with an ammonium compound, e.g. using cyano        borohydrate; and    -   d) reacting the amino group from step c) with a homobifunctional        NHS reagent in excess.

The starting substrate material having a reducing terminal sialic acidgroup used in this process should preferably have the sialic acid unitat the reducing terminal end joined to an adjacent unit through itseight carbon atom. In step b), after the reductive ring opening of theterminal sialic acid, the 6,7-diol group at the reducing end is oxidisedto form an aldehyde group at the carbon 7 atom with subsequentintroduction of amino (step c) and NHS (step d) group.

In an alternative embodiment, where the sialic acid unit at the reducingterminal end is joined to the adjacent unit through the 9 carbon atom,in step b) the C-7, C-8 diol group, at the reducing end, formed duringstep a) is oxidised to form an aldehyde group on the 8 carbon atom,followed by forming an amino group (step c) and then an NHS group (stepd).

According to one preferred embodiment, the starting material is a PSAwith a sialic acid at the reducing terminal, and also a terminal sialicacid unit at the non-reducing end which has a vicinal diol group. In thefirst step of the process (step a) a reduction reaction is performed atthe reducing end of the polysaccharide to open up the ring to furnish avicinal diol. During the reduction step the vicinal diol functionalityat the non-reducing end is not modified and remains intact. The secondstep is oxidation (step b) and during this process the vicinal diols atthe non-reducing and reducing ends will be oxidised to form aldehydegroups. In step c the aldehydes will be aminated and in step d the NHSgroups will be attached. As a result the product will be bifunctionalthat is have two NHS groups and may have useful therapeutic activitiesderived from its ability to cross-link substrates via reaction with bothNHS groups on the reducing and non-reducing end with suitablyfunctionalised substrates.

According to another preferred embodiment of the process in which thereducing terminal sialic acid is aminated, a sialic acid startingsubstrate material also having a terminal sialic acid at a non-reducingterminal end is subjected to the following steps:

-   -   e) a selective oxidation step to oxidise the non-reducing        terminal sialic acid unit at the C-7, C-8 vicinal diol group to        form a C-7-aldehyde; and    -   f) a reduction step to reduce the C-7-aldehyde group to the        corresponding alcohol.        The step also simultaneously reductively opens the sialic acid        ring on the reducing end i.e. takes place simultaneously with        step a). This aspect of the invention provides sialic acid        derivatives which have a ‘passivated’ sialic acid non-reducing        terminal, allowing activation of the reducing terminal via        periodate oxidation (step b) and reductive amination (step c).

According to a further embodiment of the invention there is provided anew process in which a sialic acid starting material having a terminalsialic acid at the non-reducing terminal end is subjected to thefollowing: step e) a selective oxidation step to oxidise thenon-reducing terminal sialic acid unit at the C-7, C-8 vicinal diolgroup to form an aldehyde on carbon atom 7; step c) conversion ofaldehyde group from step e) to an amino group by reductive aminationwith an ammonium compound and; step d) modification of the resultingamino group.

The starting material used in this embodiment of the invention shouldpreferably have the sialic acid unit at the non-reducing end joined tothe adjacent unit through the adjacent units eight carbon atom. In stepe) the C-7, C-8-diol group is oxidised to form an aldehyde group atcarbon 7 atom, that is subsequently converted to an amino group (step c)and NHS group (step d).

In an alternative embodiment, where the sialic acid unit at thenon-reducing terminal end is joined to the adjacent unit through theadjacent unit's 9 carbon atom, in step b) the C-7, C-8 diol of thisadjacent group is oxidised to form an aldehyde group on the 8 carbonatom that is replaced with an amino group, (step c) and NHS group (stepd).

The above-mentioned oxidation steps (b and e) should preferably becarried out under conditions such that there is substantially nomid-chain cleavage of a long-chain polymeric backbone starting material,and thus no substantial molecular weight reduction. Enzymes which arecapable of carrying out this oxidation step may be used. Moreconveniently the oxidation is a chemical oxidation. The reaction may becarried out with immobilised reagents such as polymer-based perruthenateor with the more straightforward method using dissolved reagents. Theoxidant is suitably perruthenate, or, preferably, periodate. Oxidationmay be carried out with periodate at a concentration in the range of 1mM to 1M, at a pH in the range 5 to 10, a temperature in the range 0 to60° C. for a time in the range 1 min to 48 hours.

Suitable reduction conditions for steps a) and step f) may utilisehydrogen with catalysts or preferably hydrides, such as borohydrides.These may be immobilised as in Amberlite supported borohydride.Preferably alkali metal hydrides such as sodium borohydride is used asthe reducing agent, at a concentration in the range 1 μM to 0.1 μM, a pHin the range 6.5 to 10, a temperature in the range 0 to 60° C. and aperiod in the range 1 min to 48 hours. The reaction conditions areselected such that pendant carboxyl groups on the PSA starting materialare not reduced. Where a preliminary oxidation step has been carried out(i.e. at the non-reducing end) the aldehyde group generated is reducedto an alcohol group which is not part of a vicinal diol group. Othersuitable reducing agents are cyanoborohydride under acidic conditions,e.g. polymer supported cyanoborohydride or alkali metalcyanoborohydride, L-ascorbic acid, sodium metabisulphite, L-selectride(trade mark), triacetoxyborohydride, etc.

During the various steps of reaction (e.g. reduction and oxidation), therespective intermediate must be isolated from oxidising and reducingagents, crosslinkers, and other reagents like NaCNBH₃, cystamine etc.,prior to being subjected to subsequent steps. Where the steps arecarried out in solution phase, isolation may be by conventionaltechniques such as expending excess oxidising agent using ethyleneglycol, ethanol precipitation, dialysis of the polysaccharide, sizeexclusion chromatography and ultrafiltration to concentrate the aqueoussolution. The product mixture from a reduction step again may beseparated by dialysis and ultrafiltration. It may be possible to devisereactions carried out on immobilised oxidising and reducing reagentsrendering isolation of product straightforward.

In the process of the invention wherein an intermediate amine compoundis produced and is then reacted with a bifunctional NHS reagent, sinceNHS groups are reactive with amine groups it is convenient to use ahomobifunctional NHS reagent. Where the two NHS groups of such a reagentare equally reactive it will be essential to use a significant excess ofthe reagent in order to minimise the extent of cross-linking, that is ofreaction of two molecules of amine intermediate with one molecule ofdi-NHS reagent. The reaction must also be carried out under conditionssuch that the second NHS group remains intact, and the reaction productmust be separable from excess unreacted NHS reagent.

NHS groups are highly unstable in water. Thus the reaction conditionsmust minimise contact of the NHS reagent with water or other proticsolvent. Preferably the reaction is carried out in dimethylsulfoxide(DMSO). It may be necessary for a small amount of water or other proticand polar solvent to be included in order to solubilise the substrate.The amount should be minimised, for instance kept below 10% of totalsolvent. It may be desirable to raise the temperature to optimisesolubilisation of the reagents and accelerate the reaction, providedthat this does not result in a chemical modification such as oxidationor cleavage of the substrate to an undesirable degree.

In a further embodiment an aldehyde terminated intermediate, producedaccording to steps a) and b) and/or step c) is reacted with hydrazine toform a hydrazone intermediate. The hydrazone groups are reactive withNHS groups. In the essential step of the process of the invention wherea bifunctional NHS reagent is reacted, the NHS reagent is conveniently adi NHS compound. The same precautions must be observed as for reactionson amine intermediates described above. A reaction scheme is shown asscheme 8 a).

Suitable di-NHS reagents are:bis[2-succinimidyloxycarbonyl-oxy)ethyl]sulfone (BSOCOES) and its sulfoanalog, bis(sulfosuccinimidyl)suberate) (BS³), disuccinimidyl glutarate(DSG), dithiobis(succinimidyl propionate) (DSP), disuccinimidyl suberate(DSS), disuccinimidyl tartrate (DST) or its sulfo analog,3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP), and ethyleneglycol bis(succinimidyl succinate) (EGS) and its sulfo analog.

According to an alternative embodiment of the process, instead of apreliminary step involving formation of an amine group on the substrate,there is provided a preliminary step in which a thiol group is provided,preferably at one terminal sialic acid unit, or, alternatively at aterminal sialic acid unit and at the terminal unit at an opposite end,which may be a sialic acid or another sugar residue.

The thiol group is, for instance, formed by reacting cystamine with analdehyde group followed by reduction. The aldehyde group may beintroduced at one or both terminal units by carrying out steps a) and b)and/or step e) on a starting material with respective terminal groups.The starting material may additionally have a non-reducing terminalsugar with a vicinal diol group which may be converted to an aldehydefor reaction with cystamine. Alternatively such a terminal group may bedeactivated by sequential oxidation then reduction steps to avoidformation of a difunctional thiol intermediate. The thiolation isconducted using the general procedure described by Pawlowski et al.

The thiol group may, alternatively be introduced in a series of stepscarried out on the amine intermediate produced above in steps a-c. Thethiol group is introduced by reaction of the amine group with a2-iminothiolane (2-IT) (Pawlowski, A. et al op. cit) comprising athiolated sialic acid unit.

A thiol group containing intermediate is a novel compound and representsa further aspect of the invention. The compound may be represented bythe following general formulae IV, V, VI, or VII

-   -   in which    -   R⁷ is a linking group;    -   A¹ is NR¹² where R¹² is H, C₁₋₄ alkyl, or aryl;    -   GlyO is a glycosyl group and k is 0-100;    -   Gly¹O is a glycosyl group which is optionally derivatised on a        pendant carboxylic acid group;    -   R⁸ is a mono-, di-, oligo- or polysaccharide group, a protein, a        peptide, a lipid, a drug, a drug delivery system, or a component        of a cell membrane or wall; and    -   R⁸ and R⁹ are each hydrogen or a mono-, di- or oligosaccharide        group, an alkyl group, an acyl group, a drug, a lipid or a drug        delivery system;    -   R¹⁰ is a mono-, di-, oligo- or polysaccharide group, a protein,        a peptide, a lipid, a drug, a drug delivery system, a component        of a cell membrane or wall or a group

-   -   R¹¹ is

-   -   wherein one of R¹³ and R¹⁴ is hydrogen and the other is a        mono-di- or oligosaccharide group, an alkyl group, an acyl        group, a drug, a lipid or a drug delivery system.

The linker group R⁷ is selected from the same groups as R² listed above.

The definitions of R⁸ and R⁹ are preferably as disclosed above aspreferred definitions for groups R³ and R⁴ respectively, GlyO ispreferably SylO.

The thiol intermediate where GlyO is SylO is reacted in the essentialstep of the process of the first aspect of the invention with aheterobifunctional linker which has a thiol-reactive functional group aswell as the NHS group. Such thiol-reactive groups are, for instanceN-maleimido groups, or thiopyridyldithio groups, vinylsulphone orN-iodoacetamine groups. Examples of suitable bifunctional reagents are:N-(α-maleimidoacetoxy)succinimide ester, (AMAS),N-(β-maleimidopropyloxy)succinimide ester, (BMPS),N-(ξ-maleimidocapryloxy)succinimide ester, (EMCS), or its sulfo analog,N-(γ-maleimidobutyryloxy)succinimide ester, (GMBS), or its sulfo analog,succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate)(LC-SMCC), m-maleimido benzoyl-N-hydroxysuccinimide ester (MBS), or, itssulfo analog,succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxyate) (SHCC) orits sulfo analog, succinimidyl-4-(p-maleimido phenyl)butyrate (SMPB) orits sulfa analog, succinimidyl-6-(β-maleimido-propionamido) hexanoate(SMPH), N-(k-maleimidoundecanoyloxy)sulfosuccinimide-ester (sulfo-KMUS),succinimidyl 6-[3-2(2-pyridyldithio)-propionamido]hexanoate (LC-SPDP) orits sulfo analog,4-succinimidyloxycarbonyl-methyl-α-(2-pyridyldithio)toluene (SMPT), orits sulfo-LC analog, N-succinimidyl-3-(2-pyridyldithio)propionate(SPDP), N-succinimidyl[4-vinylsulfonyl)benzoate (SVSB), succinimidyl3-(bromoacetamido)propionate (SBAP), and N-succinimidyliodoacetate (SIA)and N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB) or its sulfoanalog.

Reaction conditions for the reactions generally used may also be usedhere, for instance with reference to Hermanson 1995.

The above-mentioned NHS heterobifunctional reagent may be selecteddepending on its water-solubility and whether the conjugate is to becleavable or non-cleavable. The preference is for reagents with shortand non-immunogenic linkers.

The reaction with the NHS-reagent is usually to be performed in 0-100%DMSO solutions (preferably with a minimum amount of water e.g. 10%) at atemperature between 0-150° C., preferably at 20° C. The sialic acidsubstrate on the intermediate may be dissolved by aid of heat e.g.sonication or microwave preferably under inert environment. Watersoluble NHS reagents can be used when the presence of organic solventsin subsequent uses of the product cannot be tolerated. In addition,water soluble NHS-reagent may be preferred where the use of the productis for cell surface conjugation as any unreacted reagent not removedfrom the product may not permeate the cell membrane.

In the invention the starting material is a polysialic acid (PSA). Suchcompounds may comprise units other than sialic acid in the molecule. Forinstance sialic acid units may alternate with other saccharide units.Preferably, however, the polysaccharide consists substantially only ofunits of sialic acid, which preferably are alpha-2,8 and/or alpha-2,9linked (polysialic acid—hereinafter PSA).

The starting material has at least 2, preferably at least 5, morepreferably at least 10, for instance at least 50, sialic acid units. ThePSA may be derived from any source, preferably a natural source such asa bacterial, e.g. E. coli K1 or K92, group B meningococci, or even cow'smilk or N-CAM. The sialic acid polymer may be a heteropolymeric polymersuch as group 135 or group V of N. meningitidis, or may be chemicallysynthesized. The PSA may be in the form of a salt or the free acid. Itmay be in a hydrolysed form, such that the molecular weight has beenreduced following recovery from a bacterial source. The PSA may bematerial having a narrow or wide spread of molecular weights such ashaving a polydispersity of 1.01, indeed even as much as 2 or more.Preferably the polydispersity of molecular weight of PSA to be employedis less than 1.2.

A population of PSAs either in their native form, or as intermediates ofthe types described above, or the final products having a wide molecularweight distribution may be fractionated into fractions with lowerpolydispersities, i.e. into fractions with differing average molecularweights. Fractionation is preferably done by anion exchangechromatography, using for elution a suitable basic buffer. We have founda suitable anion exchange medium, namely a preparative medium such as astrong ion-exchange material based on activated agarose, havingquaternary ammonium ion pendant groups (i.e. strong base). The elutionbuffer is non-reactive and is preferably volatile so that the desiredproduct may be recovered from the base in each fraction by evaporation.Suitable examples are amines, such as triethanolamine. Recovery may beby freeze-drying for instance. The fractionation method is suitable fora PSA starting material as well other PSA derivatives. The technique maythus be applied before or after the essential process steps in thisinvention.

Preferably the process of fractionation is carried out on a populationof ionisable which have a molecular weight (Mw) preferably higher than 5kDa using ion-exchange chromatography (IEC) and using as elution buffera base or acid which is preferably volatile. Preferably the PSA hascarboxylic acid groups and the ion-exchange is anion exchange.Preferably the elution buffer contains an amine, such astriethanolamine, with recovery of PSAs preferably via freeze-drying theelution fractions.

The PSA-NHS compound of the invention may be used in a subsequentprocess for derivatising amines, e.g. of biologically useful compounds.Such reactions can be done preferably in phosphate,bicarbonate/carbonate, HEPES or borate buffers at concentrationspreferably between 5-200 mM. Other buffers may also be used if they donot contain primary amines. HEPES, for example, can be used because itcontains only tertiary amines. A large excess of Tris/Glycine at neutralto basic pH may be added at the end of the reaction to quench it. Thereactions may be preferably performed between pH 7 and 9 at 4° C. to 20°C. for 30 minutes to 2 hours. The PSA-NHS compound may be used in a 2-50fold molar excess to protein (or other derivatisable compound) dependingon the concentration of the amine. Typically, the concentration of thePSA-NHS compound may preferably vary from 0.1-10 mM. The amine, e.g.protein concentration may preferably be kept around 10-100 μM becausemore dilute protein solution result in excessive hydrolysis of the NHSgroup of the PSA-NHS compound.

The product NHS compound is of particular value for derivatising aminegroup-containing proteins, in which the amine group is suitably theepsilon amine group of a lysine or the N-terminal amino group. Theproduct is of particular value for derivatising protein or peptidetherapeutically active agents, such as cytokines, growth hormones,enzymes, hormones and antibodies or their fragments. Alternatively, theprocess may be used to derivatise drug delivery systems, such asliposomes, for instance by reacting the NHS group with an amine group ofa liposome forming component. Other drug delivery systems are describedin U.S. Pat. No. 5,846,951. Other materials that may be derivatisedinclude viruses, microbes, cells, including animal cells, and syntheticpolymers or copolymers.

The invention also provides methods in which the new compounds arereacted with biologically relevant compounds, having derivatisable aminegroups under conditions suitable for reaction of the NHS group with theamine group to form a covalent conjugate. The biologically relevantmolecule is preferably a peptide or a protein and the amine group is ona side chain of a Lys unit or is at the N-terminal of the peptide orprotein. The degree of derivatisation may be less than 1.0, but ispreferably at least 1.0, for instance at least 1.5, that is eachmolecule of biologically active compound is conjugated to at least onePSA substrate moiety.

The derivatisation of proteins and drug delivery systems by reactionwith the new PSA compounds, may result in increased half-life, improvedstability, reduced immunogenicity or antigencity, and/or control ofsolubility and hence bioavailability and pharmacokinetic properties, ormay enhance the solubility of actives or the viscosity of solutionscontaining the derivatised active.

The new preferred monofunctional PSA-NHS is highly reactive and is moreconducive to the synthesis and manufacture of a pharmaceuticallyacceptable product, since it avoids the considerable complexity whichmay be created by use of PSA forms with unmodified reducing ends (FIG.2). Production of the new form of the polymer (FIG. 5) involves,selective oxidation (step e), preferably by periodate (see earliersection) to introduce an aldehyde function at the non-reducing endfollowed by reductive amination (step c) and condensation to form NHSfunctionality (step d). Unlike the prior art illustrated in FIG. 1however, this aldehyde moiety may be destroyed by reduction (step a),for instance with borohydride. At the other end of the polymer, theborohydride reduction step also simultaneously locks open the ringstructure of the reducing end, by reducing the hemiketal. Thissimultaneous reduction of the ketone to a hydroxyl moiety introduces anew diol functionality at the reducing end, which is now amenable toselective oxidation in the second oxidation step. When the naturalpolymer has been (successively) oxidised with periodate, reduced withborohydride, oxidised a second time with periodate and aminated andconjugated to form an NHS compound, a new polymer form is created, whichis truly monofunctional, having a single reactive group (i.e. NHS group)only at the formerly reducing end (FIG. 5).

The scheme for protein reactivity by condensation of the variousderivatives is described in FIGS. 9 and 10. The monofunctional PSA cangive rise only to single-orientation attachment to proteins, with thenon-reducing end outermost, and is incapable of inadvertentlycross-linking proteins. This new scheme of reaction (FIG. 5) methodelegantly avoids the need to purify away the intended product from thevarious unintended products (described in FIG. 2), which are completelyavoided in this new reaction scheme.

The following is a brief description of the drawings.

FIG. 1a is a reaction scheme showing the prior art activation of thenon-reducing sialic acid terminal unit;

FIG. 1b is a reaction scheme showing the prior art reductive aminationof the aldehyde moiety of the product of reaction scheme 1a using aprotein-amine moiety;

FIG. 2 represents schematically the potential by-products of the sidereactions;

FIG. 3 is a reaction scheme showing the tautomerism between the ketaland ring-closed forms of the reducing terminal sialic acid unit of aPSA; In solution, the terminal sialic acid residue at the reducing endof polysialic acid exists in a tautomeric equilibrium. The ketal form,although in low abundance in the equilibrium mixture (Jennings andLugowski, 1981) is weakly reactive with protein amine groups, and cangive rise to covalent adducts with proteins in the presence of sodiumcyanoborohydride, although at a rate and to an extent that are notpractically useful.

FIG. 4 shows the preparation of reducing end derivatised NHS colominicacid (when non-reducing end has no vicinal diol)

FIG. 5 shows the preparation of reducing end derivatised NH₂-CAcolominic acid (vicinal diol removed at non-reducing end)

FIG. 6 shows the general scheme for preparation of CA-NHS-proteinconjugation

FIG. 7a shows the preparation of derivatised thiol colominic acid (CA-SHat non-reducing end)

FIG. 7b shows schematic representation of CA-protein conjugation viaCA-SH using NHS-maleimide

FIG. 7c shows the preparation of CA-protein conjugates via CA-SH usingNHS-maleimide (AMAS)

FIG. 8a shows the preparation of CA-protein conjugates via NHS onreducing end

FIG. 8b shows capping of reducing end of polysialic acid

FIG. 8c shows preparation of non-reducing end derivatised CA

FIG. 9 shows the preparation of CA-protein conjugates usingbis(sulfosuccinimidyl)suberate (BS³) on non-reducing end

FIG. 10 shows the schematic representation of CA-protein conjugationusing the crosslinker DSG

FIGS. 11a and 11b shows gel permeation chromatography (GPC)chromatograms for CAs separated as in example 5.

FIG. 12 shows size exclusion HPLC on CA-NHS-growth hormone (GH) proteinconjugation reactions (CA 35 kDa)

FIG. 13 shows the sodium dodecyl sulphate (SDS)-polyacrylamide gelelectrophoresis (PAGE) of CA-NHS-GH conjugates (CA 35 kDa)

FIG. 14 shows native PAGE of unreacted and reacted CAs

FIG. 15 shows the SDS-PAGE analysis of the CAH-NHS reactions as inexample 10

FIG. 16 shows the HPLC chromatogram of action 6 from FIG. 15.

EXAMPLES

Materials

Sodium meta-periodate and molecular weight markers were obtained fromSigma Chemical Laboratory, UK. The CAs used, linear alpha-(2,8)-linkedE. coli K1 PSAs (22.7 kDa average, polydispersity (p.d.) 1.34; 39 kDap.d. 1.4; 11 kDa, p.d. 1.27) were from Camida, Ireland. Other materialsincluded 2,4 dinitrophenyl hydrazine (Aldrich Chemical Company, UK),dialysis tubing (3.5 kDa and 10 kDa cut off limits (MedicellInternational Limited, UK); Sepharose SP HiTrap, PD-10 (Pharmacia, UK);XK50 column and Sepharose Q FF (Amersham Biosciences UK); Tris-glycinepolyacrylamide gels (4-20% and 16%), Tris-glycine sodium dodecylsulphaterunning buffer and loading buffer (Novex, UK). Deionised water wasobtained from an Elgastat Option 4 water purification unit (ElgaLimited, UK). All reagents used were of analytical grade. A plate reader(Dynex Technologies, UK) was used for spectrophotometric determinationsin protein or CA assays.

Methods

Protein and CA Determination

Quantitative estimation of CAS, as sialic add, was carried out by theresorcinol method [Svennerholm 1957] as described elsewhere [Gregoriadiset. al., 1993; Fernandes and Gregoriadis, 1996, 1997]. GH was measuredby the bicinchoninic add (BCA) colorimetric method.

Example 1—Fractionation of CA by IEC (CA, 22.7 KDa, pd 1.34) [Reference]

An XK50 column was packed with 900 ml Sepharose Q FF and equilibratedwith 3 column volumes of wash buffer (20 mM triethanolamine; pH 7.4) ata flow rate of 50 ml/min. CA (25 grams in 200 ml wash buffer) was loadedon column at 50 ml/min via a syringe port. This was followed by washingthe column with 1.5 column volumes (1350 ml) of washing buffer.

The bound CA was eluted with 1.5 column volumes of different elutionbuffers (Triethanolamine buffer, 20 mM pH 7.4, with 0 mM to 475 mM NaClin 25 mM NaCl steps) and finally with 1000 mM NaCl in the same buffer toremove all residual CA and other residues (if any).

The samples were concentrated to 20 ml by high pressure ultra filtrationover a 5 kDa membrane (Vivascience, UK). These samples were bufferexchanged into deionised water by repeated ultra filtration at 4° C. Thesamples were analysed for average molecular weight and other parametersby GPC) (as reported in example 5) and native PAGE (stained with alcianblue; example 8). Narrow fractions of CA produced using above procedurewere oxidised with sodium periodate and analysed by GPC and native PAGEfor gross alteration to the polymer.

Example 2: Activation of CA [Reference]

Freshly prepared 0.02 M sodium metaperiodate (NaIO₄; 6 fold molar excessover CA) solution was mixed with CA at 20° C. and the reaction mixturewas stirred magnetically for 15 min in the dark. The oxidised CA wasprecipitated with 70% (final concentration) ethanol and by centrifugingthe mixture at 3000 g for 20 minutes. The supernatant was removed andthe pellet was dissolved in a minimum quantity of deionised water. TheCA was again precipitated with 70% ethanol and then centrifuged at12,000 g. The pellet was dissolved in a minimum quantity of water,lyophilized and stored at −20° C. until further use.

Example 3: Determination of the Oxidation State of CA and Derivatives[Reference]

Quantitative estimation of the degree of CA oxidation was carried outwith 2,4 dinitrophenylhydrazine (2,4-DNPH), which yields sparinglysoluble 2,4 dinitrophenyl-hydrazones on interaction with carbonylcompounds. Non-oxidised CA and oxidised CA (CAO) (5 mg each) were addedto the 2,4-DNPH reagent (1.0 ml), the solutions were shaken and thenallowed to stand at 37° C. until a crystalline precipitate was observed[Shriner et. al., 1980]. The degree (quantitative) of CA oxidation wasmeasured with a method [Park and Johnson, 1949] based on the reductionof ferricyanide ions in alkaline solution to ferric ferrocyanide(Persian blue), which is then measured at 630 nm. In this instance,glucose was used as a standard.

Example 4a: Preparation of Amino Colominic Acid (CA-NH₂) [Reference]

CAO at 10-100 mg/ml was dissolved in 2 ml of deionised water with a300-fold molar excess of NH₄Cl, in a 50 ml tube and then NaCNBH₄ (5 Mstock in 1 N NaOH(aq), was added at a final concentration of 5 mg/ml.The mixture was incubated at room temperature for 5 days. A controlreaction was also set up with colominic acid instead of CAO. Productcolominic acid amine derivative was precipitated by the addition of 5 mlice-cold ethanol. The precipitate was recovered by centrifugation at4000 rpm, 30 minutes, room temperature in a benchtop centrifuge. Thepellet was retained and resuspended in 2 ml of deionised water, thenprecipitated again with 5 ml of ice-cold ethanol in a 10 mlultracentrifuge tube. The precipitate was collected by centrifugation at30,000 rpm for 30 minutes at room temperature. The pellet was againresuspended in 2 ml of deionised water and freeze-dried.

Example 4b: Assay for Amine Content

The TNBS (picrylsulphonic acid, i.e. 2,4,6-tri-nitro-benzene sulphonicacid) assay was used to determine the amount of amino groups present inthe product [Satake et. al., 1960].

In the well of a microtitre plate TNBS (0.5 μl of 15 mM TNBS) was addedto 90 μl of 0.1 M borate buffer pH 9.5. To this was added 10 μl of a 50mg/ml solution of CA-amine. The plate was allowed to stand for 20minutes at room temperature, before reading the absorbance at 405 nm.Glycine was used as a standard, at a concentration range of 0.1 to 1 mM.TNBS trinitrophenylates primary amine groups. The TNP adduct of theamine is detected.

Testing the product purified with a double cold-ethanol precipitationusing the TNBS assay showed close to 85% conversion.

Example 4c: Preparation of Colominic Acid—SH

Oxidised CA was derivatised with cystamine by reductive amination asdescribed in example 4a, except using a 100-fold molar excess ofcystamine instead of NH₄Cl.

Before purifying the product, it was treated with 50 mM DTT at 37° C.for 1 h. The reduced product was purified by double ethanolprecipitation and size exclusion chromatography on sepharose G25.

In another example, CANH₂ prepared as in example 4a, is dissolved in 10mM PBS with 1 mM EDTA pH 8.0. A 50-fold molar excess of 2-iminothiolaneis added and the reaction allowed to proceed for 1 h at 25° C. Unreacted2-iminothiolate is removed by gel filtration on a sephadex G25 columnequilibrated with the reaction buffer.

Thiol content is estimated using the Ellman's assay. Briefly 150 ofsample are mixed with 150 μl of 0.1M phosphate, 1 mM EDTA, pH8containing 0.08 mg/ml 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) andallowed to react for 30 minutes at room temperature and read at 405 nm.The product is suitable for reaction according to the schemes in FIGS.7b and 7 c.

Further, the thiol content of the polymer was found to be 60%.

Example 4d: Preparation of CA-NHS

CA-NH₂ (35 kDa) (15-20 mg) synthesised in Reference Example 4a above wasdissolved in 0.15M PBS (3504 pH 7.2) and then either 50 or 75 molarequivalents of BS³ in PBS (150 PH 7.2) was added. The mixture wasvortexed for 5 seconds and then reacted for 30 minutes at 20° C. TheCA-NHS product was purified by PD-10 column using PBS as eluent (pH 7.2)and used immediately for site-specific conjugation to the NH₂ groups inproteins and peptides. Determination of the CA concentration from the PD10 fractions was achieved by analysing the sialic acid content using theresorcinol assay. The NHS content on the CA polymer was measured by UVspectroscopy by analysing the CA and NHS reaction solution at 260 nm andalso by thin layer chromatography with visualization at 254 nm.

CA-NH₂ (35 kDa) (15-20 mg) synthesised in Example 4a above was eitherdissolved in the minimum amount of water (50-65 0) to which was addedDMSO (300-235 μL) or in >95% DMSO (350 with the aid of heat (100-125°C.). 75 molar equivalents of DSG in DMSO (150 L) was added to the CA-NH,solution, vortexed for 5 seconds and then reacted for 30 minutes at 20°C. The CA-NHS product was purified either with dioxane precipitation(×2) or by PD-10 column using PBS as eluent (pH 7.2) and usedimmediately for site-specific conjugation to the NH₂ groups in proteinsand peptides. As before determination of the CA concentration from thePD-10 fractions was measured using the resorcinol assay. The NHS contenton the CA polymer was measured by UV spectroscopy (260 nm) and by thinlayer chromatography (254 nm).

Example 5: Gel Permeation Chromatography of CA Samples [Reference]

CA (35 kDa) samples were dissolved in NaNO₃ (0.2M), CH₃CN (10%; 5 mg/ml)and were chromatographed on 2×GMPW_(XL) columns with detection byrefractive index (GPC system: VE1121 GPC solvent pump, VE3580 R1detector and collation with Trisec 3 software (Viscotek Europe Ltd).Samples (5 mg/ml) were filtered through 0.45 μm nylon membrane and runat 0.7 cm/min with 0.2M NaNO₃ and CH₃CN (10%) as the mobile phase (FIG.11).

Example 6: Preparation of CA-NHS-Protein Conjugates (Using BS³ and DSG)

GH in sodium bicarbonate (23 mg/ml, pH 7.4) was covalently linked toCA-NHS (35 kDa), from example 4b using an excess of BS³. The reactionwas performed in 0.15 M PBS (pH 7.2; 1.5 ml) using a molar ratio of 25:1or 50:1 of CA-NHS:GH for a period of 30 minutes at 20° C. PolysialylatedGH was characterised by SDS-PAGE and the conjugation yield determined byHPLC-size exclusion chromatography. Controls included subjecting thenative protein to the conjugation procedure using BS³ in the absence ofany CA-NHS. CA-NH₂ was also subjected to the conjugation procedure usingBS³ in the absence of native GH.

GH in sodium bicarbonate (pH 7.4) was covalently linked to CA-NHS (35kDa), which was prepared as discussed in example 4b using an excess ofDSG. The reaction was performed in 0.15 M PBS (pH 7.2; 1.5 ml) using amolar ratio of 50:1 of CA-NHS:GH for a period of 30 minutes at 20° C.Polysialylated GH was characterised by SDS-PAGE and the conjugationyield determined by HPLC-size exclusion chromatography. Controlsincluded subjecting the native protein to the conjugation procedureusing DSG in the absence of any CA-NHS.

Example 7: HPLC-SEC of CA-NHS-GH Conjugates

CA-GH conjugates were dissolved in ammonium bicarbonate buffer (0.2M;pH7) and were chromatographed on superose 6 column with detection by UVindex (Agilent, 10/50 system, UK). Samples (1 mg/ml) were filtered over0.45 μm nylon membrane 175 μl injected and run at 0.25 cm/min withammonium bicarbonate buffer as the mobile phase (FIG. 12).

Example 8: SDS and Native PAGE of CAs and CA-GH Conjugates

SDS-PAGE (MiniGel, Vertical Gel Unit, model VGT 1, power supply modelConsort E132; VWR, UK) was employed to detect changes in the molecularsize of GH upon polysialylation. SDS-PAGE of GH and its conjugates (withCA-NHS) at 0 minutes (control) and 30 minutes samples from the reactionmixtures as well as a process control (non oxidised CA), was carried outusing a 4-20% polyacrylamide gel. The samples were calibrated against awide range of molecular weight markers (FIGS. 13 and 14).

Results

CA (22.7 kDa) and its derivatives were successfully fractionated intovarious narrow species with a polydispersity less than 1.1 with m.w.averages of up to 46 kDa with different % of populations. Table 2 showsthe results of separating the 22.7 kDa material.

TABLE 2 Ion exchange chromatography of CA22.7 (pd 1.3) Elution buffers(in 20 mM Triethanolamine buffer + mM NaCl, pH 7.4) M.W. Pd % Population325 mM 12586 1.091 77.4%  350 mM 20884 1.037 3.2% 375 mM 25542 1.0145.0% 400 mM 28408 1.024 4.4%  425 mM* 7.4% 450 mM 43760 1.032 2.3% 475mM 42921 1.096 0.2% *Not done

This process was scalable from 1 ml to 900 ml of matrix with thefractionation profile almost identical at each scale (not all resultsshown).

The fractionation of larger polymer (CA, 39 kDa, pd 1.4) producedspecies up to 90 kDa. This process can successfully be used for thefractionation of even large batches of the polymer. The results showthat the on exchange fractions are narrowly dispersed. This isconsistent with the GPC data.

All narrow fractions were successfully oxidised with 20 mM periodate andsamples taken from different stages of the production process andanalysed by GPC and native PAGE, which showed no change in the molecularweight and polydispersity.

CA, a PSA, is a linear alpha-2,8-linked homopolymer ofN-acetylneuraminic acid (Neu5Ac) residues (FIG. 1a ).

Quantitative measurement of the oxidation state of CA was performed byferricyanide on reduction in alkaline solution to ferrocyanide (PrussianBlue) [Park and Johnson, 1949] using glucose as a standard. The oxidizedCA was found to have a nearly 100 mol % of apparent aldehyde content ascompared to native polymer. The results of quantitative assay of CAintermediates in the oxidation process using ferricyanide wereconsistent with the results of qualitative tests performed with DNPHwhich gave a faint yellow precipitate with the native CA, and intenseorange colour with the aldehyde containing forms of the polymer,resulting in an intense orange precipitate after ten minutes of reactionat room temperature.

The integrity of the internal alpha-2,8 linked Neu5Ac residues postperiodate and borohydride treatment was analysed by GPC and thechromatographs obtained for the oxidised (CAO), amino CA (CA-NH₂),CA-NHS materials were compared with that of native CA. It was found(FIG. 12) that all CAs exhibit almost identical elution profiles, withno evidence that the various steps give rise to significantfragmentation or crosslinking (in case of CA-NHS) of the polymer chain.The small peaks are indicative of buffer salts.

Formation of the CA-GH conjugates was analysed by SEC-HPLC and SDS-PAGE.For the conjugation reaction with DSG the SDS-PAGE showed that there wasno free GH remaining and that the conjugation reaction had gone tocompletion. This was confirmed by SEC-HPLC, whereby the CA-GH conjugateswere eluted before the expected elution time of the free GH (a peak forfree GH was not observed). On the other hand, analysis by SDS-PAGE ofthe conjugation reaction of CA-NH₂ to GH using BS³ showed the presenceof free GH, which was confirmed by SEC-HPLC with an elution peak around70 minutes for the free protein. In addition, the SEC-HPLC enable thedegree of conjugation to be determined at 53%.

The results (FIG. 13) show that in the conjugate lanes there are shiftsin the bands which typically indicates an increase in mass indicative ofa polysialylated-GH in comparison to GH. Further, GH conjugates wereseparated into different species by SEC-HPLC.

Example 9: Preparation of Colominic Acid Hydrazide (CAH) [Reference]

50 mg of oxidised colominic acid (19 kDa) was reacted with 2.6 mg ofhydrazine (liquid) in 400 μl of 20 mM sodium acetate buffer, pH 5.5, for2 h at 25° C. The colominic acid was then precipitated with 70% ethanol.The precipitate was redissolved in 350 μl phosphate buffer saline, pH7.4 and NaCNBH₃ was added to 5 mg/ml. The mixture was allowed to reactfor 4 h at 25° C., then frozen overnight. NaCNBH₃ and reaction byproducts were removed by gel permeation chromatography on a PD10 columnpacked with Sephadex G25, using 0.15M NH₄HCO₃ as the mobile phase. Thefractions (0.5 ml each) were analysed by the TNBS assay (specific toamino groups; described earlier). Fractions 6, 7, 8 and 9 (the voidvolume fractions) had a strong signal, well above the background. Thebackground was high due to the presence of the NH₃ ions. Fractions 6, 7,8 and 9 also contained colominic acid. These four fractions, were freezedried to recover the CA-hydrazide (CAH).

Example 10: Preparation of Colominic Acid NHS (CA-NHS) and ColominicAcid-Protein Conjugates

10 mg of 19 kDa CA-hydrazide were reacted with 9 mg of BS³ in 400 μl ofPBS (pH 7.4) for 30 minutes at room temperature. The reaction mixturewas applied to a PD-10 column packed with Sephadex G25 collecting 0.5 mlfractions. 0.1 mg of BSA was added to each fraction between 5 and 9.After 2 hours at room temperatures the fractions reacted with BSA. Thesesamples were analysed by SDS-PAGE and SEC HPLC.

These fractions have little colominic acid. The colominic acid richfractions (6 and 7) have a protein streak in addition to the bandspresent in the other samples and BSA, which is clear evidence ofconjugation (FIG. 15).

The HPLC chromatogram of fraction 6 shows that there is a big shift inthe retention time for conjugate as compared to free protein confirmingconjugation (FIGS. 16a and b ).

The BSA used contains impurities. The BSA peak is at 56 minutes (FIG.16a ).

In addition to peak at 56 minutes, there are larger species which areconjugates. There is a large peak at 80 minutes, which is the NHSreleased from the CA-NHS as it reacts with the protein. This cannot befree BS as the CAH was passed through a gel permeation chromatographycolumn, which will have removed it. This strongly suggests that an NHSester group was created on the CA molecule (FIG. 16b ).

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The invention claimed is:
 1. A process of making an activated polysialicacid (PSA) molecule comprising a terminal sialic acid unit at thereducing end, the process comprising: a) reducing the terminal sialicacid unit at the reducing end to form a vicinal diol group; b)selectively oxidizing the vicinal diol group of step (a) to form analdehyde group; c) aminating the aldehyde group of step (b) to form anamine group; and d) reacting the amine group of step (c) with abifunctional reagent comprising two amine-reactive functional groups toform an activated PSA molecule, the activated PSA molecule beingmonofunctional and comprising an amine-reactive group at the reducingend.
 2. The process of claim 1, wherein the PSA molecules consistingsubstantially only of units of sialic acid alpha-2,8 PSA moleculesand/or alpha-2,9 linked PSA molecules.
 3. The process of claim 1,wherein the PSA molecule comprises at least 5 sialic acid units.
 4. Theprocess of claim 3, wherein the PSA molecule comprises at least 10sialic acid units.
 5. The process of claim 4, wherein the PSA moleculecomprises at least 50 sialic acid units.
 6. The process of claim 1,wherein in step (a) the reducing agent used is a hydride or hydrogenwith catalysts.
 7. The process of claim 6, wherein the hydride is analkali metal hydride.
 8. The process of claim 1, wherein in step (a) thereducing agent used is, an alkali metal cyanoborohydride, L-ascorbicacid, sodium metabisulphite, or triacetoxyborohydride.
 9. The process ofclaim 1, wherein in step (a) the terminal sialic acid unit at thereducing end is joined to an adjacent unit through its 8 carbon atom andthe vicinal diol group formed is a 6,7-diol group.
 10. The process ofclaim 9, wherein in step (b) the 6,7-diol group is oxidized to form analdehyde group at carbon atom
 7. 11. The process of claim 1, wherein instep (a) the terminal sialic acid unit at the reducing end is joined toan adjacent unit through its 9 carbon atom and the vicinal diol groupformed is a 7,8-diol group.
 12. The process of claim 11, wherein in step(b) the 7,8-diol group is oxidized to form an aldehyde group at carbonatom
 8. 13. The process of claim 1, wherein in step (b) the selectiveoxidation is performed using an enzymatic oxidation process or achemical oxidation process.
 14. The process of claim 1, wherein in step(b) the selective oxidation is carried out using perruthenate, orperiodate.
 15. The process of claim 1, wherein each of the twoamine-reactive functional groups comprises an unsubstituted succinimidylgroup or a substituted succinimidyl group.
 16. The process of claim 15,wherein the substituted succinimidyl group is an ester ofN-hydroxysuccinimide.
 17. The process of claim 1, wherein thebifunctional reagent is selected from the group consisting ofbis[2-succinimidyloxycarbonyl-oxy)ethyl]sulfone (BSOCOES) or a sulfoanalog thereof, bis(sulfosuccinimidyl)suberate) (BS³), disuccinimidylglutarate (DSG), dithiobis(succinimidyl propionate) (DSP),disuccinimidyl suberate (DSS), disuccinimidyl tartrate (DST) or a sulfoanalog thereof, 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP),and ethylene glycol bis(succinimidyl succinate) (EGS) or a sulfo analogthereof.
 18. The process of claim 1, wherein the activated PSA moleculeformed in step (d) is general formula I or II

wherein R¹ is H or sulfonyl; R² is a linking group; A is NR⁵, NR⁵N R⁶, Oor SR wherein R⁵ and R⁶ are independently selected from H, C₁₋₄ alkyland aryl; SylO is a sialyl group; n is 1-100; R³ is H, a monosialic acidgroup, a disialic acid group, an oligosialic acid group or a polysialicacid group; and R⁴ is H, a monosialic acid group, a disialic acid group,an oligosialic acid group, a polysialic acid group, an alkyl group or anacyl group.
 19. The process of claim 18, wherein R² is alkanediyl,arylene, alkarylene, heteroarylene, or alkyl-heteroarylene, any of whichis optionally interrupted by either thioester, ester, amine or amidelinkages, and in which A is NR⁵ or a linker group joined to the rest ofthe molecule through a group NR⁵.
 20. The process of claim 18, wherein Ais NR⁵, wherein R⁵ is H.
 21. The process of claim 18, wherein R²comprises an alkane-diyl group together with a carbonyl to which A isattached.
 22. The process of claim 21, wherein R² is C_(p)H_(2p)CO₃wherein p is 2-12.
 23. The process of claim 18, wherein R² comprises analkanediyl group with an alkane carbon atom attached to A.
 24. Theprocess of claim 18, wherein R² comprises a mid-chain ester, an amide,ether, a thioether and/or a 1-thio-N-succinimidyl amine linkage.
 25. Theprocess of claim 18, wherein R² comprises an alkyleneoxyalkylene or analkyleneoligooxyalkylene.
 26. The process of claim 18, wherein

is a member selected from the group consisting of


27. The process of claim 1, wherein when the PSA molecule comprises atthe non-reducing end a terminal sialic acid having a vicinal diol group,the process further comprises: e) selectively oxidizing the vicinal diolgroup of the non-reducing terminal sialic acid unit to form an aldehydegroup; f) aminating the aldehyde group of step (e) to form an aminegroup; and g) reacting the amine group of step (f) with a bifunctionalreagent comprising two amine-reactive functional groups to form theactivated PSA molecule, the activated PSA molecule being bifunctionaland comprising an amine-reactive group at the reducing end and anamine-reactive group at the non-reducing end.
 28. The process of claim27, wherein in step (e) the non-reducing terminal sialic acid unit isjoined to an adjacent unit through its 8 carbon atom and a 7,8-diolgroup and the aldehyde formed is an aldehyde group at carbon atom
 7. 29.The process of claim 27, wherein in step (e) the non-reducing terminalsialic acid unit is joined to an adjacent unit through its 9 carbon atomand a 7,8-diol group and the aldehyde formed is an aldehyde group atcarbon atom
 8. 30. The process of claim 27, wherein in step (e) theselective oxidation is performed using an enzymatic oxidation process ora chemical oxidation process.
 31. The process of claim 27, wherein instep (e) the selective oxidation is carried out using perruthenate, orperiodate.
 32. The process of claim 27, wherein each of the twoamine-reactive functional groups comprises an unsubstituted succinimidylgroup or a substituted succinimidyl group.
 33. The process of claim 32,wherein the substituted succinimidyl group is an ester ofN-hydroxysuccinimide.
 34. The process of claim 27, wherein thebifunctional reagent is selected from the group consisting ofbis[2-succinimidyloxycarbonyl-oxy)ethyl]sulfone (BSOCOES) or a sulfoanalog thereof, bis(sulfosuccinimidyl)suberate) (BS³), disuccinimidylglutarate (DSG), dithiobis(succinimidyl propionate) (DSP),disuccinimidyl suberate (DSS), disuccinimidyl tartrate (DST) or a sulfoanalog thereof, 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP), orethylene glycol bis(succinimidyl succinate) (EGS) or a sulfo analogthereof.
 35. The process of claim 27, wherein the activated PSA moleculeformed in step (d) is general formula Ill

wherein R¹ is H or sulfonyl; R² is a linking group; A is NR⁵, NR⁵N R⁶, Oor SR wherein R⁵ and R⁶ are independently selected from H, C₁₋₄ alkyland aryl; SylO is a sialyl group; and m is 0-100.
 36. The process ofclaim 35, wherein R² is alkanediyl, arylene, alkarylene, heteroarylene,or alkyl-heteroarylene, any of which is optionally interrupted by eitherthioester, ester, amine or amide linkages, and in which A is NR⁵ or alinker group joined to the rest of the molecule through a group NR⁵. 37.The process of claim 35, wherein A is NR⁵, wherein R⁵ is H.
 38. Theprocess of claim 35, wherein R² comprises an alkane-diyl group togetherwith a carbonyl to which A is attached.
 39. The process of claim 38,wherein R² is C_(p)H_(2p)CO₃ wherein p is 2-12.
 40. The process of claim35, wherein R² comprises an alkanediyl group with an alkane carbon atomattached to A.
 41. The process of claim 35, wherein R² comprises amid-chain ester, an amide, ether, a thioether and/or a1-thio-N-succinimidyl amine linkage.
 42. The process of claim 35,wherein R² comprises an alkyleneoxyalkylene or analkyleneoligooxyalkylene.
 43. The process of claim 35, wherein

is a member selected from the group consisting of


44. A method of conjugating a biological molecule to an activated PSAmolecule, the method comprising conjugating an activated PSA molecule ofclaim 1 by reacting with a biological molecule comprising an aminegroup.
 45. The method of claim 44, wherein the biological molecule isselected from the group consisting of a protein, a peptide, a drug, alipid, a liposome, a microbe, a cell or component thereof, a syntheticpolymer or a synthetic copolymer.
 46. The method of claim 45, whereinthe protein is selected from the group consisting of a cytokine, anenzyme, a hormone, an antibody or a fragment thereof.
 47. A method ofconjugating a biological molecule to an activated PSA molecule, themethod comprising conjugating an activated PSA molecule of claim 27 byreacting with a biological molecule comprising an amine group.
 48. Themethod of claim 47, wherein the biological molecule is selected from thegroup consisting of a protein, a peptide, a drug, a lipid, a liposome, amicrobe, a cell or component thereof, a synthetic polymer or a syntheticcopolymer.
 49. The method of claim 48, wherein the protein is selectedfrom the group consisting of a cytokine, an enzyme, a hormone, anantibody, or a fragment thereof.